System for enhanced longevity of in situ microbial filter used for bioremediation

ABSTRACT

An improved method for in situ microbial filter bioremediation having increasingly operational longevity of an in situ microbial filter emplaced into an aquifer. A method for generating a microbial filter of sufficient catalytic density and thickness, which has increased replenishment interval, improved bacteria attachment and detachment characteristics and the endogenous stability under in situ conditions. A system for in situ field water remediation.

The United States Government has rights in this invention pursuant toContract No. W-7405-ENG-48 between the U.S. Department of Energy and theUniversity Of California, for the operation of Lawrence LivermoreNational Laboratory.

This application is a division of application Ser. No. 08/706,152, filedAug. 20, 1996, now U.S. Pat. No. 5,888,395.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention concerns a method and a system for improved in situbioremediation of groundwater by increasing the operational longevity ofan in situ microbial filter emplaced in an aquifer. The longevity of thebiofilter is increased by selecting a stable bacterial isolate andenhancing its longevity with one or more additives. In particular, thisinvention provides a method for producing a microbial filter ofsufficient catalytic density and thickness, and for increasing thebacterial replenishment interval, via improved attachment and detachmentcharacteristics of the cells and their endogenous catalytic stabilityunder the devised in situ attachment conditions.

Attachment and detachment characteristics of the bacteria of theinvention are enhanced by screening an otherwise pure homogeneouspopulation of a single bacterial strain for stable isolates, preferablyrosette cluster forming isolates, and improving these properties bymodifications of the injection cell buffer.

The bioremediation system of the invention comprises a bioreactor forgrowing a biofilter of bacterial cells, a biofilter, a means foremplacement of the biofilter in situ in a contaminated water aquifer anda means for extracting remediated water through the biofilter.

2. Background and Related Art

Groundwater aquifers at many industrial, research and defense-relatedsites are often contaminated with chlorinated volatile organic compounds(VOCs). While "pump and treat" is currently the standard method forremediating these compounds, in situ bioremediation using bacterialfilters described, for example, in Geomicrobiol., 8:133-146 (1991),continues to generate much interest.

Methanotrophic bacteria, containing the soluble form of methanemonooxygenase (sMMO), are known to oxidatively degrade a number ofchlorinated aliphatic hydrocarbons, including trichloroethylene (TCE)(Appl. Environ. Microbiol., 57:228-235 and 1031-1037 (1991)). A specificin situ microbial filter approach described in Hydro Sci. J., (IAHS),38:323-342 (1993) utilizes resting-state (nondividing) cells of themethanotroph, Methylosinus trichosporium OB3b. In this filter strategy,cells that have been previously grown in a bioreactor are injected intothe subsurface ahead of a dilute, migrating contaminant plume. Ideally,as the contaminated groundwater flows into this zone, the attachedmicrobial population degrades the contaminant(s) at a rate that keepspace with the rate of transport, and the groundwater then exits clean.However, an in situ pilot field test of a M. trichosporium OB3b filter,used for a narrow, shallow, fast moving TCE plume at a contaminated siterevealed that the microbial filter had only short term longevity(several days), in terms of an efficient removal of TCE, and therefore,very limited utility in groundwater remediation (Environ. Sc. Technol.,30: 1982-1989 (1996). Need for frequent replacement of the filterbiomass makes this treatment method uneconomical and impractical.

To be economically viable and operationally achievable in the field, thein situ microbial filter will have to degrade TCE and several otherchlorinated ethanes over a period of several weeks before it isreplenished. Several parameters influence the filter's ability toachieve this: the finite biotransformation capacity of the attachedresting cells for contaminants, the attachment/detachment properties ofthe injected bacteria with respect to the natural geological sedimentsor an introduced in situ sand trench and the long term endogenousstability of the whole-cell sMMO activity. Without improved and enhancedfunctional or operational longevity, the whole-cell rate of hydrocarboncontaminant catalysis and the biotransformation capacity would decay toorapidly to extremely low values over time. This, in turn, would prove tobe impractical for in situ bioremediation applications.

Therefore, it would be desirable to provide a laboratory-basedpredictive methodology for creating an economical bacterial filter at afield site, a biofilter having an improved and enhanced longevity thatwould allow a continuous remediation without a need to replace themicrobial filter more than once in about 6-8 weeks.

Because bacteria are particles, the colloid filtration theory provides abasis for studies of cell transport through saturated, subsurface media.Advection, dispersion, deposition, and entrainment are all processesthat can influence this movement (Geomicrobiol., 8:133-146 (1991)). Yetoverall, it is generally accepted that cell surface hydrophobicity andcell/solid electrostatic interactions are the most important factorswhich influence bacterial deposition onto, and retention by, aquifersediments. Experimentally, physical properties (e.g., grain size) aswell as chemical properties (ionic strength and pH) have been shown toinfluence the attachment density of bacteria to aquifer sands andmineral surfaces. Generally, an ionic strength of ˜0.01 molal is neededto promote maximal attachment, but the process is not electrolytespecific. Additionally, chemical alterations of both the aqueous andsolid media and of the bacterial cell surface can be quite important.For example, it has been reported that Mg and Fe oxide coatings ofaquifer sands increase bacterial attachment (J. Contam. Hydrol.6:321-336 (1990).

While the need for improved microbial filter remediation of contaminatedgroundwater persists and while attempts at providing a means for suchimproved remediation have been made, due to a short lifespan ofavailable microbial filters, such remediation efforts have not beensuccessful.

It would be, therefore, advantageous to provide validatable methods andsimulated conditions that would permit an increased longevity of anyspecific bacteria that might be used for an in situ microbial filter,especially as it relates to enhancing their attachment and detachmentproperties.

It is therefore a primary objective of this invention to utilize theresting-state M. trichosporium OB3b cells or other bacteria havingimproved attachment and detachment properties as an in situ biofilterfor the treatment of ground water containing a chlorinated hydrocarboncontaminant, such as a TCE-contaminated plume.

All patents, patent applications and publications cited herein areincorporated by reference.

SUMMARY

One aspect of this invention is an improved in situ validatablebioremediation method for sites contaminated with hydrocarbons ingeneral and with chlorinated aliphatic hydrocarbons, in particular.

Another aspect of this invention is a validatable bioremediation methodusing an emplaced microbial filter having an improved and enhancedlongevity due to increased attachment and decreased detachmentproperties.

Still another aspect of this invention is a microbial filter useful forin situ remediation wherein the microbial filter is created using astable isolate of an otherwise pure strain of M. trichosporium OB3b,wherein the stable isolate is selected by initial pre-screening of a M.trichosporium OB3b culture for the isolate able to form rosetteclusters.

Another aspect of this invention is a modified medium allowing a growthof bacterial strains having an improved and enhanced longevity due toincreased attachment and decreased detachment properties.

Still another aspect of this invention is a modified suspension mediafor bacterial strains having an improved and enhanced longevity due toincreased attachment and decreased detachment properties.

Yet another aspect of this invention is an improved bioremediationmethod comprising using an emplaced microbial biofilter having aincreased half-life to about and over 8 weeks.

Still yet another aspect of this invention is a bioremediation systemcomprising a bioreactor for growing a sufficient biomass of bacterialcells to form an emplaced biofilter, a biofilter, a means foremplacement of the biofilter in situ in contaminated water aquifer, anda means for extraction of water through the biofilter.

Another aspect of this invention is a laboratory method for predicting,designing and optimizing conditions for field water remediation takinginto consideration the field conditions, such as a type of acontaminant, pH, temperature, soil type, dissolved oxygen and inherentproperties of the bacterial strain or its substrain.

Still yet another aspect to this invention is a method forbioremediation of contaminated groundwater by biodegradation ofcontaminants using an emplaced microbial biofilter, said methodcomprising steps:

(a) selecting a bacterial strain able to biodegrade a water contaminantto be emplaced as a microbial biofilter for the biodegradation of thewater contaminant using a laboratory method for predicting, designingand optimizing conditions for bioremediation according to the invention;

(b) cultivating said bacterial strain to a cell biomass having increasedlongevity by increasing the attachment and decreasing detachment rate ofthe biofilter;

(c) emplacing said microbial biofilter at a site of a contamination; and

(d) extracting the contaminated groundwater through the microbialbiofilter;

wherein the bacterial strain is selected from the group of bacteria suchas methanotrophic bacteria possessing an enzyme such as oxygenase orenzymatic system such as monooxygenase system able to biodegrade thecontaminant, and wherein such bacteria is preferably a rosette clustersforming isolate of Methylosinus trichosporium OB3b strain;

wherein the bacteria is pregrown on nitrate minimal salt medium lackingcopper to the cell biomass having a density necessary to biodegrade thecontaminant for at least 8 weeks;

wherein said cell biomass is emplaced as the biofilter in an aquifer bysuspending the cell biomass in an injection medium substituted withadditives such as magnesium salts, ferrous salts and agar.

Still yet another aspect of the current invention is a system forbioremediation of contaminated water comprising components:

(a) a means including a laboratory method for identification of stablebacterial isolates able to form rosette clusters and their separationfrom the isolates not having such ability;

(b) a surface bioreactor for growth of sufficient biomass of selectedisolate;

(c) a mixing chamber wherein the cells or cell paste of the selectedisolate is suspended in water and additives are added;

(d) an injection apparatus for the biofilter formation in situ;

(e) a microbial biofilter emplaced in situ;

(f) a means for extraction of water through the biofilter; and

(g) a means for monitoring purity of the remediated water;

wherein the bioreactor further contains a means for controlling thetemperature, pH, nutrients supply and oxygen or other gas needed forbacterial growth;

wherein preferably the injection apparatus is an injection pump and thecell suspension is injected through existing injection or extractionwells into the aquifer to form an emplaced attached biofilter having thedensity and thickness to assure a longevity and functionality of thebiofilter for at least about 8 weeks.

Still another aspect of the current invention is a laboratory method forprediction, design and optimization of conditions for fieldbioremediation said method comprising steps:

(a) determining pH, dissolved oxygen, temperature and soil type of acontaminated site aquifer;

(b) determining an identity of a contaminant and a degree ofcontamination;

(c) selecting a bacteria able to biodegrade said contaminant for anemplaced biofilter;

(d) determining attachment/detachment properties of the selectedbacteria under the conditions of step (a);

(e) designing an injection buffer composition and additives foroptimization of the biofilter emplacement, its longevity andfunctionality;

(f) confirming the designed conditions optimization withattachment/detachment assays followed with metabolic assays;

wherein said selection method of step (c) comprises comparing rosetteforming isolates among themselves as well as comparing them to singlecell isolates using the attachment assay, submitting all investigatedisolates to a treatment with a single additive or a combination ofadditives, testing said isolates in the detachment assay, selecting themost stable isolate and determining the isolate's longevity.

Still yet another aspect to the current invention is a biofiltersuitable for emplacement into a contaminated groundwater aquifer forbiodegradation of a water contaminant comprising a bacterial cellbiomass wherein said bacteria are able to biodegrade the contaminant andthe biofilter has a longevity at least 8 weeks;

wherein preferably the bacterial cell biomass comprises a bacterialstrain selected from the group of bacteria possessing an enzyme such asan oxygenase or enzymatic system such as a monooxygenase system able tobiodegrade the contaminant;

wherein the bacteria is preferably a Methylosinus trichosporium OB3brosette clusters forming isolate.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A is a schematic for the creation of an in situ biofilter in theintermediate subsurface region surrounding a single vertical well.

FIGS. 1A and 1B show the actual field data using the bioractor method.

FIGS. 2A, 2B, and 2C are a schematic of an in situ bioremediation methodthat depends on an improved microbial filter, which is created andimplemented in three different configurations FIGS. 2A, 2B, and 2C.Configuration C involves the emplacement of a sand trench.

FIG. 2D shows a line-of-wells including those of FIG. 2A, 2B, and 2C.

FIG. 3 is an illustration of the laboratory apparatus for the simulatedattached-cell functional longevity testing and validation.

FIGS. 4A and 4B depict micrographs of a single-cell suspension (FIG. 4A)and a rosette suspension (FIG. 4B), both derived from the same otherwisepure strain culture of M. trichosporium OB3b.

FIGS. 5A, 5B, and 5C are a schematic of bacterial attachment/detachmentmeasurements by a sand-column pumping procedure.

FIGS. 6A and 6B are graphs showing attachment of the M. trichosporiumOB3b rosette (colony 5) and a single cell (colony 2) isolates as afunction of the loading time (FIG. 6A) and cell density (FIG. 6B).

FIGS. 7A and 7B are graphs showing detachment profiles of the M.trichosporium OB3b rosette (colony 5) (FIG. 7A) and the single cell(colony 2) (FIG. 7B) isolates as a function of time following cellloading with different buffer mixtures.

FIGS. 8A through 8L inclusive, are graphs showing the degradation ofweekly TCE pulses for 15 weeks by the rosette forming isolate M.trichosporium OB3b.

FIGS. 9A and 9B are plots of the weekly profiles of the non-metabolizedTCE (A) and the bacterial detachment (B) for the attached Cell TestColumn in FIG. 8.

DEFINITIONS

As used herein:

"Methanotrophs or methanotrophic" means an aerobic bacteria that havethe ability to obtain both their energy and their carbon for theirgrowth by oxidizing methane gas. Some methanotrophs when preciselycultured in the absence of copper contain exclusively the soluble formof methane monooxygenase.

"sMMO" means soluble form of methane monooxygenase. "Contaminant" or"contaminants" means different types of contaminants currently known tobe readily susceptible to bacterial biodegradation or biotransformation,such as petroleum and coal-derived hydrocarbons and their derivatives,halogenated aliphatics including trichloroethene (TCE), halogenatedaromatic and nitroaromatics. These contaminants may be transformed byeither aerobic or anaerobic bacterial processes, as described inMicrobiological Reviews, 55:59-79 (1991).

"Biofilter", "filter", "attached cell filter or biofilter" or "microbialfilter or biofilter" means a mass of bacteria, preferably methanotrophicbacteria emplaced in the aquifer and able to biodegrade the volatileorganic contaminants (VOCs). The biofilter is also meant to encompassother types of bacteria such as bacteria possessing oxygenase enzymesystems within several genera of aerobic microorganisms able tocometabolize TCE in an aerobic process. Examples of these bacteria arePseudomonas cepacia G4 possessing the toluene-orthomonooxygenase systemand Pseudomonas putida F1 possessing the toluene dioxygenase system.Other candidates for the biofilter are several different types ofindigenous bacteria, such as Pseudomonas, Arthrobacter, Mycobacterium,and Rhodococcus, which can use a variety of polycyclic aromatichydrocarbons (PAHS) as a source of carbon and energy; anaerobicbacteria, such as Clostridium bifermentans CYS1 which anaerobicallydegrades high explosives such as trinitrotoluene (TNT),hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), andoctahydro-1,3,5,7-tetraazocine (HMX) to non toxic aliphatic endproducts; Pseudomonas sp. strain KC able to transform carbontetrachloride (CT) to carbon dioxide, formate, and nonvolatile endproducts; and other anaerobic microorganisms such as for example,Desulfovibrio vulgaris capable of the reductive biotransformation ofmetals such as for example Cr(Vl), Fe(III), Mn (IV), Se (VI), to theirless toxic reduction products.

"Rosette forming" or "rosette isolate" means a substrain of an otherwisepure homogenous strain of M. trichosporium OB3b6 which can be isolatedas a separate colony by agar plating, but when it is repeatedly culturedit propagates mainly as clusters of cells, as opposed to a single cellform.

"Attachment" means chemical or physical bacterial association with sandor other natural subsurface sediments after a standard loading method.

"Detachment" means bacteria eluting with time from sand or other naturalsubsurface sediments after the standard attachment assay.

"Additives" means any compounds or a mixture thereof added either to thegrowth medium for growing a cell biomass or any compound added to aninjection buffer for a biofilter emplacement. Typically, the additiveswill include media and agars used for culturing the bacterial cellswhich are commercially available and which can be used unsupplemented orsupplemented with other additives. The additives also means variousbuffers suitable for growth of bacterial cells or for their maintenance.These buffers would typically have pH from about 4.3 to about 8.5,preferably from about 7.0 to 7.8 and most preferably from about 7.4 to7.6. Additives also include salts, minerals, vitamins, enzymes catalystsand any other compound which will promote growth and stability of thebacterial cell isolates, increase their attachment or decrease theirdetachment. Exemplary medium is Higgins' nitrate minimal salts mediumlacking Cu. Exemplary buffer is a phosphate buffer. Exemplary salts arenitrous, ferrous, nickel, molybdenum, potassium, sodium, calcium, copperand zinc chlorides, sulfates, phosphates as well as oxides and othercompounds. Additives are meant to encompass any and all compounds whichwill aid in the method of the invention and promote the cells growth andstability.

"Higgins'" or "Higgins' phosphate buffer" or "HPB" means 0.01 Mphosphate buffer (pH 7.5) used in the culture medium to grow thebacteria, M. trichosporium OB3b.

"Modified Higgins' salts medium" or "(MHS)" means Higgins' nitrateminimal salts medium (NMS) described in Biotechnol. Bioeng., 38:423-433, (1991) prepared by doubling the nitrate and Fe concentrationsto 20 mM and 80 μm, respectively, raising the concentration of Na₂MoO₄.2H₂ O 40-fold to 16 μm, and adding 7.5 μM NiCl₂ 6H₂ O according toHydrological Sci. J., 38: 323-342 (1993).

"Validatable" means able to be validated.

"VOC" means volatile organic compounds such as benzene, toluene, hexane,xylene and others.

"Resting cells" means metabolically active, but non-dividing bacteria.

DETAILED DESCRIPTION OF THE INVENTION

The current invention provides a method and a system for improved insitu water bioremediation using microbial filter having extendedfunctional longevity, emplaced in a contaminated aquifer. The methodinvolves creating the microbial filter in situ by selecting a stableisolate of an appropriate pure bacterial strain, increasing thebacterial isolate's density and the attachment properties and decreasingthe detachment rate of the isolate cells, thereby increasing theoperational longevity of an in situ emplaced microbial filter.

The filter is preferably formed of methanotrophic aerobic bacteria thathave the ability to obtain both their energy and their carbon for growthby oxidizing methane gas. Methanotrophs when cultured in the absence ofcopper have been found to contain exclusively the soluble form ofmethane monooxygenase. Isolates of other bacteria, such as those listedin definitions may be similarly used if they possess the samebiodegradative activity toward the contaminant(s) of interest and ifthey can be cultured is a stable form.

The method generates a microbial filter of sufficient catalytic densityand thickness having improved cell attachment and detachmentcharacteristics, wherein the acceptable overall operational longevity ofthe microbial filter is extended from a few days to about and preferablyover 8 weeks.

Attachment and detachment characteristics of the bacteria of theinvention are enhanced by the selection of the microbial strain isolateor its substrain which is able to form rosettes isolate and bymodification of the injection cell buffer. The bioremediation methodaccording to the invention is enhanced by the parallel development ofquantitative and predictive assays that simulate attachment/detachmentand operational longevity performance in the field which allowlaboratory testing of these features before the method is applied in thefield.

The bioremediation method is economical as well as practical and safe.The economic feasibility of the in situ microbial filter is dependent onits operational longevity, which in turn is dependent on several keyparameters. Among these parameters are the bacterial attachmentdensities reached during the injection of the microbial suspension andthe subsequent detachment-removal of cells from the filter over time.

Briefly, according to the invention, in situ microbial bioremediation ofaquifers contaminated with chlorinated aliphatic VOCs or othercontaminants is achieved with an in situ biofilter using pregrown,resting microbial cells able to form cluster rosettes or otherwisechange their attachment/detachment properties resulting in enhancedlongevity.

The in situ methanotrophic filter strategy in this invention utilizespregrown resting cells, such as M. trichosporium OB3b isolated as asubstrain of the original strain. These cell are identified and selectedbecause of their ability to be cultured indefinitely in a form havingenhanced attachment properties, such as ability to be cultured in arosette cluster form. These cell are then grown on a suitable inorganicsalts medium promoting the growth of the selected cells strains orsubstrains, or on a modified medium, such as for example medium lackingcertain metal component, preferably copper, in case of M. trichosporiumOB3b. These conditions should be such that the attachment of the cellsor cell clusters suspensions to wet saturated sand is increased byapproximately 2-fold versus that of the parent strain or single cellnon-cluster suspensions.

The attachment then is further increased another 2-fold by anappropriate modification of the cell or cluster suspension loadingbuffer, preferably by addition of a magnesium salt, ferrous salt anddilute agar. These modifications in the medium and buffer result infurther increasing the attachment half-life of the microbial biofiltergenerated using rosette-clusters or other cells having enhancedattachment properties. The half-life and the longevity of the biofilteris extended by slowing the time-dependent detachment rate from about 1week to and over 8 weeks.

Improvements and the quantitative assays developed for the purposes ofthis invention and described herein make the microbial filterremediation feasible for decontamination of large volumes of flowingunderground aquifers. Using the method of the invention, thecontaminants present in the aquifer are biodegraded by microbialcometabolic action to unharmful components and, consequently, theaquifer water may be used or discharged without need for furtherprocessing and purification.

I. Microbial Biofilter Remediation

The microbial biofilter remediation according to the invention and thesuccess of a resting cell biofilter strategy in the field is dependenton creating a microbial filter of sufficient catalytic density andthickness so that the residence time of the filter in situ provides thedesired amount of contaminant degradation before the filter needs to bereplenished. The utility and economic feasibility of such emplacedmicrobial filter depends on the microbial biomass replenishment intervalwhich should be at least about 8 weeks, or preferably longer.

The replenishment interval depends on

1) the biotransformation capacity of the cells for the contaminant;

2) the attachment/detachment characteristics of the bacteria; and

3) the endogenous stability of the enzymatic system cometabolizing thecontaminant of the attached whole-cell.

In case of M. trichosporium OB3b, the replenishment interval depends onthe stability of the soluble methane monooxygenase (sMMO) enzyme systemunder the in situ filter conditions.

The method and the system for the microbial biofilter remediation areillustrated in FIGS. 1-9.

A. Method for Microbial Filter Remediation

The invention in one aspect concerns a method for bioremediation ofcontaminated groundwater by biodegradation of the contaminant using anin situ emplaced microbial biofilter.

Typically, the method of the invention comprises selecting a stableisolate of the bacterial strain able to biodegrade a water contaminantin the laboratory and determining its properties, i. e. its longevity,vis-a vis a water contamination and the degree of the contamination. Theselected isolate is cultivated to a cell biomass needed to create anemplaced biofilter having increased longevity to at least but preferablyover 8 weeks by increasing the attachment and decreasing the detachmentrate of the biofilter. The cell biomass is then emplaced in situ as amicrobial biofilter for the biodegradation of the water contaminant. Thebiofilter is typically emplaced in aquifer by suspending the cellbiomass in an injection medium substituted with selected additivespromoting its growth, stability, attachment and detachment. Typically,the injection medium is substituted with magnesium salts, ferrous saltsand with agar added in concentration from about 0.0075% to about 0.025%on a dry weight/0.01 M phosphate buffer (HPB) volume basis. Thecontaminated groundwater is extracted through the microbial biofilter bytypically pumping the water out of the contamination site through thefilter.

Implementation of the resting-cell in situ microbial biofilter accordingthe invention, utilizing as exemplary bacteria a cluster form of M.trichosporium OB3b, is seen in FIGS. 1 and 2.

FIG. 1A is a schematic flow-chart of the present in situ biofilterremediation method and a system. In practice of the invention, thebiofilter bacteria, preferably methanotrophs, are first grown forseveral weeks in a large surface bioreactor (Step 1) in a nitrateminimum salt medium, preferably Higgins' medium lacking copper, attemperature from about 20° C. to about 35° C., preferably under amethane/air gas mixture. Rosettes populations are then separated fromthe medium according to the procedure of the Example 1. Growth medium isthen discarded. The rosette cluster forming cells are used for thebiofilter formation. These cells are preferably reduced to paste, andtransported to the bioremediation site (Step 2). During transportationthe cells are preferably chilled. The cells or cell paste are mixedand/or evenly suspended in water supplemented with additives such asmagnesium and ferrous salt and agar at the site of contamination (Step3) and the cell suspension is injected (Step 4) through the well, suchas cell injection or groundwater-extraction well, into the aquifer toform an emplaced attached biofilter (arrow). Remediation via themicrobial filter proceeds in situ (Step 5) by extracting thedecontaminated groundwater through the attached biofilter. The extractedwater is tested for residual contaminants.

The actual field data using the bioreactor method obtained in fieldtesting are shown in FIGS. 1A and 1B. As seen in insets 1 and 2,virtually complete removal of the initial contamination (background inFIGS. 1A) is accomplished. Groundwater originally containing about 430part-per-billions (ppb) contaminants (FIG. 1A), is remediated to containless than 10 ppb of contaminant (FIG. 1B).

In the remediation biofilter method, further illustrated in FIGS. 2A,2B, 2C, and 2D, the attached-cell microbial filter can be configured andemplaced in any one of several ways. The filter can be emplaced at thebeginning of the plume as a cylindrical zone around a single injectionwell also known as a Huff-and-Puff emplacement (FIG. 2A); as a linearwall created with a line-of-wells for injection and withdrawal, innatural heterogenous subsurface media (FIG. 2B); or as a linear wallproduced within an introduced homogenous sand filled trench (FIG. 2C)via a line-of-wells positioned in the saturated sand as seen in FIG. 2D.Contaminated water from any one of these well configurations is pumpedthrough the filter in the direction of groundwater flow and plumemigration. The filter cometabolizes the contaminant(s) anddecontaminated water is run through monitoring wells as seen in FIG. 2D.

Regardless of its configuration, the success of the attached biofiltersystem in the field is dependent on creating a biofilter of sufficientcatalytic density and thickness so that the residence time of the filterin situ gives the desired amount of contaminant degradation. Contaminantdegradation is a capability of the attached biofilter to achieve acontaminant biotransformation. Biotransformation capacity of themicrobial filter is determined by the attachment of the biofilter to theaquifer sand or rock, by the detachment characteristics of the bacteria,and by the endogenous stability of soluble methane monooxygenase enzymesystem of the attached biofilter cells under the in situ filterconditions.

Improving any of these parameters increases the longevity of thebiofilter and decreases the cost of operating the filter by lengtheningthe attached bacterial replenishment interval.

This invention, therefore, concerns the discovery that the biofiltermade with rosette clusters of the selected bacteria, rather than singlecells in suspension, and then treated with additives in a special wayduring the biofilter creation, extends the operational utility time ofthe filter from about 1 week up to about and over 8 weeks.

B. Microbial Biofilter

The invention provides a microbial biofilter suitable for long-termemplacement into a contaminated groundwater aquifer for biodegradationof a water contaminant. The biofilter is created by emplacing a cellbiomass of a selected stable isolate of the bacteria able to biodegradethe contaminant. The biofilter biomass and properties of the bacterialisolate extend the biofilter's longevity to at least but preferably over8 weeks.

The biofilter typically is made of the isolate of a bacterial strainpossessing an enzyme or enzymatic system able to biodegrade a specificcontaminant. For most harmful and common underground water contaminants,such as hydrocarbons, the biofilter utilizes a bacterial strainpossessing an oxygenase or monooxygenase system, such as methanemonooxygenase. The parent bacteria possessing such an enzymatic systemare methanotrophic. Representative of these bacteria on which theinvention was tested and developed is Methylosinus trichosporium OB3bstrain. This strain has now been shown to contain a rosette clustersforming isolate which has an extraordinary ability to surviveunderground and to cometabolize the contaminants for extended periods oftime.

The biofilter of the invention is preferably the Methylosinustrichosporium OB3b stable isolate, pregrown on nitrate minimal saltmedium lacking copper to the cell biomass having a density necessary toextend a half-life of the biofilter for at least 8 weeks.

The microbial biofilter of the invention utilized in the subsurfacebioremediation method of the invention thus consists of the biomass ofattached bacterial cells clusters having specific metabolicbiodegradative capabilities allowing metabolic degradation of thecontaminants of concern. Examples of suitable bacteria are M.trichosporium and other methanotrophs, and a number of other bacterialisted in definitions which are useful for the oxidation or otherchemical biodegradation of chlorinated aliphatic hydrocarbons or othercontaminants as listed in the definitions. However, other non-listedbacteria possessing similar biodegradative properties under theconditions and in the system of this invention are also intended to bewithin the scope of this invention.

The biofilter is created by first selecting an appropriate general purestrain of bacteria suitable for degradation of specific contaminants onthe basis of their metabolic properties and emplaced within the aquifer.The typical procedure for selection of bacteria is to identify thecontaminant as being a metabolic substrate for a selected bacterialstrain. It has now been discovered that an otherwise pure strain ofbacteria can be screened further, in a simple manner previouslyunrecognized, for even more suitable substrains possessing morepronounced metabolic characteristics in connection with any contaminantcandidate to be subjected to in situ microbial filter bioremediation.Such bacteria are particularly screened for increased attachmentproperties combined with a decreased or slowed rate of detachment.

A desirable isolate can have these properties due to a variety ofreasons. For example, for selection of M. trichosporium substrain OB3bfor biodegradation of VOCs, the preliminary selection criterion was theability to biodegrade the VOCs inherent in the parent strain, and thesecondary criterion was the OB3b substrain's ability to form clusters ofrosettes. Other attributes that enhance attachment, and hence are usefulas substrain pre-screening criteria, include increased size,electrostatic charge or some other property that confers or increasescell surface stickiness to the bacteria.

The selected isolate, like the original strain, is then grown in abioreactor until the biomass needed to form in situ biofilter is reachedand that biomass is then injected into the subsurface.

The injection buffer in which the cells are suspended prior to injectionto the aquifer is modified to further enhance the attachment and to slowdetachment of the biofilter. For example, the additives enhancing theattachment and slowing of detachment of M. trichosporium OB3b were agarand ferrous and magnesium salts. Different additives might beappropriate for other bacteria or subsurface condition. Any bacteriumthat can attach to the subsurface soil or sediments of interest (e.g.s,sand, clay silicate, and carbonate based materials) and has thenecessary metabolic properties to effect the type of contaminantbioremediation of concern is a potential candidate for this type ofsubstrain-selection enhancement. The method is generally applicable toall contaminants which may be biodegraded by the bacterial cells.

C. A Laboratory Method for Prediction, Design and Optimization ofConditions for a Field Bioremediation

In another aspect, the bioremediation method includes, is complementedand its effectivity is enhanced with a newly developed laboratory methodfor prediction, quantitation, design and optimization of conditions forfield bioremediation.

The laboratory method involves determining pH, amount of dissolvedoxygen, temperature and soil type and composition as well as adetermination of identity of a contaminant and a degree of contaminationof a contaminated site aquifer. Based on the above information, anappropriate bacterial strain possessing metabolic capabilities tocometabolize the contaminant is selected for an emplaced biofilter. Suchselection consists of testing attachment properties of the individualisolates of the selected bacterial strain with or without addition of avariance of additives described elsewhere. After the optimal conditions,additives and amounts thereof for the growth and stability of the testedisolates are determined, the isolates are tested for their detachmentproperties and the best and most stable isolate is then selected, itslongevity determined and if the half-life longevity is found to be aboutor longer then 8 weeks, the isolate is used for the emplaced biofllter.

The laboratory method also involves designing of an injection buffercomposition and additives for optimization of conditions for thebiofilter emplacement, its longevity and functionality. Typically, theattachment assay is followed with optimization of the additives, such asvarious salts, preferably magnesium or ferrous salts and agar, added toan injection medium. The optimization of the injection buffer or mediumis followed with detachment assay. All isolates are tested and resultsof the attachment and detachment assays and additives optimization isanalyzed and the most stable isolate is selected. Stability of theisolate depends on the length of the attachment which should beincreased as much as possible and on the length of the detachment whichshould be decreased as much as possible. The most stable isolates arethose which have the longest attachment time and the shortest detachmenttime.

After the selection of the best isolate, the isolate is tested for itshalf-life longevity by challenging the isolate cometabolic ability withvarious contaminants and different concentrations thereof. Preferably,the longevity should be about 15 weeks. Most preferably, the isolate'scometabolic activity is challenged with water from the site of acontaminated aquifer.

A critical factor in deciding whether bioremediation is possible for acontaminant at a specific site is whether the microorganism to be usedis compatible with the specific conditions and/or characteristics, suchas pH, dissolved oxygen, temperature, soil type, a type of contaminant,etc., of the contaminated site.

An overall laboratory pre-screening process able to determine and tovalidate the attachment and detachment properties of the biofilter wasdeveloped in the process of this invention.

Typically, after determining the contaminant of the aquifer and theconditions and characteristics of the contaminated site, selecting anappropriate bacterial strain or substrain possessing required metabolicactivity as a biofilter bacteria and, if necessary, modifying theinjection buffer for bacterial biofilter with additives, a series ofattachment/detachment assays followed by metabolic assays using -sitespecific sand or rock is carried out to determine the optimal injectionconditions for remediation of that particular site.

The initial bacterial attachment rate determines the amount of biomassneeded to create a filter of a given catalytic density and thickness,while the detachment rate contributes to the frequency of replenishment.These rates are determined by assay according to the Example 2. Knowingthe above parameters, i.e. attachment and detachment rate allows thecurrent method to be generalized in that a resting-cell microbialbiofilter may be designed in such a way that its initially emplacedcatalytic density, transformation capacity, and attached biomassprovides a near complete (i.e. to <5 ppb) or a maximal extent ofcontaminant degradation.

Additionally, the method allows a safety net by allowing anoverengineering. For example, the biofilter can be engineered in such away that even if the biofilter's catalytic activity would decreasemarkedly (e.g. 90%), it would still meet its flow-throughTCE-degradation design limit. This overengineering of the filter lessensa frequency of its replacement.

The invention, therefore, concerns a method for prediction, design andoptimization of conditions for field bioremediation said methodcomprising steps:

(a) determining pH, dissolved oxygen, temperature and soil type of acontaminated site aquifer;

(b) determining an identity and degree of a contaminant;

(c) identifying a bacteria able to biodegrade said contaminant for anemplaced biofilter and testing its individual isolates for theirstability and suitability as the emplaced biofilter;

(d) determining attachment/detachment properties of the tested isolateunder the conditions of step (a);

(e) designing an injection buffer composition and additives foroptimization of the biofilter emplacement, its longevity andfunctionality;

(f) confirming the designed conditions optimization withattachment/detachment assays followed with metabolic assays; and

(g) selecting the isolate having the highest stability and attachmentand lowest detachment properties.

An illustration showing the apparatus and complete experimental set-upfor conducting the field simulatory column-filter experiments appears inFIG. 3. The laboratory apparatus seen in FIG. 3 is suitable for thesimulated attached-cell functional longevity testing and validation ofthe bioremediation method as well as for the optimization of conditionsfor the fields remediation. The laboratory apparatus includes thesyringe pump (20), syringes for adding the TCE-phenol red (PR) pulses(24), the glass 1-cm×10-cm horizontal columns (28), the stainless-steelconnectors (30), and the screw-capped 5-mL fraction collection vials(32).

D. A System for Biofilter Remediation

The invention also concerns a system for bioremediating contaminatedwater. The system for remediation typically consists of at least asurface bioreactor for growth of sufficient biomass of selectedbacterial isolates, a mixing chamber wherein the isolate's cells or cellpaste is suspended in water and additives are added, an injectionapparatus for the biofilter formation in situ, a microbial biofilteremplaced in situ, a means for extraction of water through the biofilter.Additionally, the system typically contains a means for monitoring apurity of the remediated water and a means for controlling thetemperature, pH, nutrients supply and oxygen or other gas needed forbacterial growth.

The system's injection apparatus is typically a pump which injects thecell suspension through existing injection or extraction wells into theaquifer to form an emplaced attached biofilter. The system's waterextraction is typically achieved via an extraction pump placed in anextraction well. The purity of decontaminated water is determined usinga monitoring well or laboratory equipment able to detect thecontaminant.

The first component is a large surface bioreactor for growth of theselected bacteria until the sufficient biomass of bacteria to form aneffective biofilter is obtained. The bioreactor typically has a meansfor controlling the temperature, pH, nutrients supply and oxygen orother gas needed for bacterial growth.

The second component of the system is a means for separation of the cellclusters culture from the growth medium. This component can be anysuitable separator such as centrifuge. Once the bacteria are grown, theyare separated from the growth medium, and the selected bacteria aretransported to the site of the contamination.

The third component of the system is a mixing chamber where the cells orcell paste is suspended in water and transferred to a next component ofthe system, namely to the injection component for the biofilterformation in situ.

The fourth component of the system is an injection apparatus orpreferably pump injecting the cell suspension using preferably the onsite existing injection and/or extraction wells having configuration asseen in FIG. 2. There, the cell suspension is injected through the wellinto the aquifer to form an emplaced attached biofilter.

The fifth component of the system is the microbial biofilter emplaced insitu. The biofilter has preferably the density and thickness appropriateto the degree of contamination to assure the longevity of the functionalbiofilter for at least about 8 weeks.

The sixth component of the system is a means for extraction of waterthrough the biofilter utilizing any suitable means, such as extractionpumps or extraction wells.

The final component of the system is a means for monitoring theremediated water, such as a monitoring well and laboratory equipmentable to detect a degree of contamination. E. M. trichosporium OB3bBiofilter.

M. trichosporium OB3b contains a sufficient level of soluble methanemonooxygenase (sMMO) to degrade cometabolically a number of chlorinatedaliphatic hydrocarbons, including the highly toxic and regulatedcontaminant TCE. When cultured under the conditions of this invention,two distinct populations of M. trichosporium OB3b were found as shown inFIG. 4. one population consisted of single cells, the second populationconsisted of the rosette clusters. In one of the attributes of theinvention, it was subsequently found that the M. trichosporium OB3brosette isolate when subcultured successively for months does not looseits ability to grow in this rosette cluster form and is thereforesuitable for large scale productions. The rosette clusters population ofM. trichosporium OB3b was therefore a suitable candidate to test themethod of the invention for the bioremediation of water contaminatedwith TCE.

For this purpose, M. trichosporium OB3b, a rosette-cluster formingisolate, was pregrown and then further screened by quantitative assaysdeveloped to assess its attachment, detachment, and functional metaboliclongevity in a flow-through sand column.

FIGS. 4A and 4B are phase-contrast micrographs showing two populationsof M. trichosporium OB3b, derived from the same otherwise pure strainculture of M. trichosporium OB3b. M. trichosporium OB3b, as a singlecell isolate suspension, is seen in FIG. 4A and the rosette-formingisolate is seen in FIG. 4B. Corresponding buffer suspensions of theparental strain culture are dominated by single non-clustered bacteriaand microscopically appear to be the same as the cells in FIG. 4A. Thecluster bacteria form seen in FIG. 4B clearly show a pattern ofaggregation or clustering of the cells.

In both cases, the separately cultured, washed cells were suspended in10 mM Higgins' phosphate buffer pH 7.0 (HPB). The microscopicmagnification was 1,200-fold.

The single-cell and the rosette isolates were selected by pickingindividual colonies from agar spread plates. They were then maintainedas separate stock subcultures apart from the original parental strainculture, based on their stable perpetuation of either a single-cellpopulation (colony 2) (FIG. 4A) or a mostly rosette containingpopulation (colony 5) (FIG. 4B) upon continued suspension culturing. TheM. trichosporium OB3b rosettes are consistent with the rosette-likeclusters typically seen in various type II methanotrophic cultures(Appl. Environ. Microbiol., 59:2380-2387 (1993).

A form of M. trichosporium OB3b (colony 5) that microscopically appearsto exist predominantly in rosette clusters was further cultured andstudies were conducted to ascertain differences between the rosetteisolate and the single cell isolate of the original parental strain inthe attachment to or detachment from saturated sand particles.Additionally, various chemical additives were tested to enhance theattachment density and to retard the detachment rate of the clustersfrom this sand. Specifically, pretreating the rosette-isolate with acombination of magnesium and iron salts and dilute agar was found togreatly enhance its attachment density to a saturated quartzitic sand.This was not observed for the single cell form that typifies theparental strain.

Subsequently, it has been demonstrated experimentally with aflow-through sand column that an attached resting cell methanotrophicbiofilter, prepared with the rosette cells, operates extremely well forat least 2 months and to a lesser degree up to 4 months. Moreover, thisbiofilter continues to display significant chlorinated aliphatic VOCdegradative activity even after 4 months.

To establish and maintain an in situ biofilter, it is crucial tomaximize the cell attachment obtainable during the subsurface injectionof the resting cells and to slow the detachment. The use of the rosettecontaining isolate of M. trichosporium OB3b, as compared to using thesingle cell form, markedly increased the attachment density.

Using the general protocol depicted in FIG. 5A, 5B, and 5C severalparameters for the attachment of colony 5 and colony 2 to saturated sandwere examined.

Specifically, for attachment phase measurements, columns (1 cm×10 cm)were prepared with Oklahoma No.1 sand in 10 mM Higgins phosphate HPBbuffer having pH 7.0. Bacteria were pulled into columns at 0.33mL/minute for 2 hours. The column was equipped with a peristaltic pump.Unattached bacteria were washed out at 0.11 mL/minute for 16 hours.Then, the sand and attached bacteria were extruded from the columns andthe attached bacteria were enumerated. As depicted in FIG. 5C, after the2 hours cell loading step, the column tubings and the position of theperistaltic pump are switched. Following the 16 hour 10 mM HPB cellwashout step, and columns are rotated 90 degrees to a horizontalposition, the flow rate is reduced, and fractions are collected duringthe subsequent detachment phase, also using HPB alone.

FIGS. 6A and 6B illustrate the attachment of M. trichosporium OB3bcolony 5 and colony 2 to 1 cm×10 cm saturated sand columns as a functionof the loading time and cell density. In FIG. 6A cells at 2×10⁹ /mL wereloaded for the various times shown before the unattached bacteria werewashed out for 16 hours, as outlined above. In FIG. 6B the cell densitywas varied, but at each concentration the bacteria were loaded for 2hours. The attachment results plotted represent the averagevalues±standard deviation for at least three independent determinationsfor each data point.

FIGS. 6A and 6B show that the attachment of M. trichosporium OB3b in HPBexhibited a saturation kinetics type of behavior with respect to boththe loading time and the loading cell density, irrespective of whichcolony was used. Both cell types showed similar profiles. Yet, colony 5reached a maximal attachment density of ˜8×10⁸ cells/g dry sand, whilecolony 2 yielded only ˜4×10⁸ cells/g dry sand under standardtime-density attachment conditions (2 hours, 2×10⁹ cells/mL loadingdensity).

Effect of the loading-buffer mixture on the attachment of M.trichosporium OB3b is summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Effects of the Loading-Buffer Mixture                                         on the Attachment of M. trichosporium OB3b                                                       Attachment - cells/g                                       Bacterial suspension.sup.b                                                                       sand × 10.sup.8                                      loading-buffer mixture.sup.a                                                                     Colony 5 Colony 2                                          ______________________________________                                        10 mM HPB alone     6.9 ± 0.2                                                                          4.2 ± 0.1                                      10 mM HPB + 1.0 mM MgCl.sub.2                                                                     8.6 ± 1.2                                                                          3.2 ± 0.1                                      10 mM HPB + 100 μM FeSO.sub.4                                                                  6.3 ± 0.8                                                                          3.8 ± 0.4                                      10 mM HPB + 0.075% agar.sup.c                                                                    12.3 + 1.8                                                                             4.3 ± 0.2                                      10 mM HPB + 0.025% agar.sup.c                                                                    13.5 ± 1.4                                                                          5.6 ± 0.8                                      10 mM HPB + 1.0 mM MgCl.sub.2 +                                                                  14.3 ± 0.8                                                                          5.1 ± 0.9                                      100 μM FeSO.sub.4 + 0.0075% agar                                           10 mM HPB + 1.0 mM MgCl.sub.2 +                                                                  14.3 ± 0.8                                                                          5.1 ± 0.9                                      100 μM FeSO.sub.4 + 0.025% agar                                            ______________________________________                                         .sup.a Bacteria were suspended in the indicated buffer mixture and loaded     onto 1cm × 10cm columns                                                 .sup.b The data listed represent the average values ± standard             deviation for three independent determinations                                .sup.c The agar included in the loading buffer was purified agar prepared     on a dry weight/volume basis in HPB.                                     

Table 1 shows the effects of the changes in the bacterial loading buffermixture on the attachment of colony 5 and colony 2 under standardtime-density conditions. The attachment of colony 2 was unaffected bythe addition of magnesium or ferrous salts to the loading buffer, whilethe addition of 0.025% agar increased its attachment by 1.3-fold. Colony5 attachment also was not influenced by FeSO₄, but was enhanced1.25-fold by 1 mM MgCl₂, and 2.0-fold by 0.025% agar. These effects onthe colony 5 cells were additive since Mg+Fe+0.025% agar improved itsattachment by 2.3-fold to a maximal density of 15.6×10⁸ bacteria/g drysand.

Pasteur pipet attachment experiments in which such disposable mini-glasscolumns were scored with a file and then segmented, indicated that thisincreased colony 5 attachment was not due to entrainment on the tops ofthe columns (data not shown).

To maintain an in situ biofilter and to extend its longevity, it is alsoimportant to maximize the detachment retardation. This applies if in thebiofilter creation it has been overengineered by initially emplacing amuch higher density of attached cells than might be needed.

HPB detachment profiles for colony 5, previously loaded with the variousbuffer mixtures in Table 1, are plotted in FIG. 7A. HPB detachmentprofiles for colony 2 suspensions loaded with two of the buffer mixturesare shown in FIG. 7B.

Specifically, for detachment phase measurements, columns (1 cm×10 cm)were prepared with Oklahoma No.1 sand in 10 mM HPB buffer having pH 7.0.Bacteria were pulled into columns at 0.33 mL/minute for 2 hours asoutlined in FIG. 5. After the 2 hour cell loading step, the columntubing and the position of the peristaltic pump were switched.Unattached bacteria were washed out at 0.11 mL/minute for 16 hours. Thecolumns were then rotated 90° to a horizontal position, the HPB flowrate was reduced to 2.0 mL/h (6.25 cm/hour linear flow rate) andfractions were collected and bacteria were enumerated.

FIG. 7A, 7B shows the detachment profiles of M. trichosporium OB3bcolony 5 and colony 2, respectively, with 10 mM HPB alone, as a functionof time following cell loading with several different buffer mixtures.For cell suspensions that had been loaded only in the presence of 10 mMHPB, the detachment half-lives were 5 days and ˜1 day, for colony 5 andcolony 2, respectively. The inclusion of 1 mM MgCl₂ or 100 μM FeSO₄ inthe loading buffer decreased the detachment rate of colony 5 onlyslightly. However, the addition of very dilute concentrations of agar tothe loading buffer markedly slowed its detachment. The detachmenthalf-life of colony 5 in the presence of 0.0075% of agar and 0.025% agarwere increased to 10 and 32 days, respectively (FIG. 7A). The inclusionof 1 mM MgCl₂ and 100 μM FeSO₄ with the HPB-buffered 0.0075% or 0.025%agar further retarded the colony 5 detachment, to half-lives of 13 and45 days, respectively. The detachment half-life for colony 2 remainedabout the same in the presence of 1 mM MgCl₂ +100 μM FeSO₄ +0.025% agar,namely ˜1-1.5 days; although, a slow tailing by 5-10% of these cells wasevident in its detachment profile (FIGS. 7A and 7B).

After the initial 2 hour load step, both the 16 hour wash out buffer andthe detachment was HPB. Similar detachment profiles to those seen inFIG. 7A for colony 5 were obtained when carbonate-based groundwater fromtwo different sites were substituted for HPB as the detachment solution(data not shown). Thus, the greatly enhanced detachment half-livespromoted by MgCl₂ +FeSO₄ +dilute agar are specific to colony 5 versuscolony 2, but they are not confined to a single electrolyte such asphosphate.

Microscopic examination confirmed that during the 2 hour loading phasein the presence of 10 mM HPB-buffered MgCl₂ +FeSO₄ and 0.0075% or 0.025%agar, colony 5 tended to aggregate into larger rosette clusters, whilecolony 2 remained as a single cell population. Aggregation of colony 5did not occur, even after many hours in HPB alone. Agar atconcentrations above 0.025% caused plugging of the sand columns bycolony 5 and, therefore, would not in general be recommended. Theadvised range of dilute agar to incorporate into the cell loading(laboratory columns) or cell injection (field site) buffer mixture wouldbe from about 0.0075% to about 0.025% on a dry weight/HPB volume basis.

Also critical to the enhanced attachment of the bacteria was the diluteagar included in the cell loading (injection) buffer. Since agar is amixture of polysaccharides and may mimic the holdfast material, itseffect on the loading buffer was tested. Dilute agar proved to be themajor contributor to an increased attachment (Table 1) and the retardeddetachment (FIG. 7A), perhaps due to its ability to cause the rosettesof colony 5 to aggregate into somewhat larger groups. Alternately, theagar in the cell loading mixture may also aid attachment by neutralizingthe sand surface silicates in combination with the Fe⁺² and Mg⁺²cations. Irrespective, since colony 2 does not aggregate in the samemanner, there seems to be an intrinsic difference in the chemicalconstitution of the surfaces of these two isolates.

When selecting additives to be included with the loading buffer, it isimportant to take advantage of any peculiarities of the particularbacterial system chosen. In the case of the M. trichosporium OB3brosette isolate, agar (a mixture of polysaccharides) was chosen to mimicthe polysaccharide-containing material that holds the rosettes together.

In designing the conditions and selection of additives, it is importantto take into account any special characteristics of the additives sothat the additives do not have any adverse effects on the whole-celldegradative activity of the bacteria. This should be verified beforeusing them in the attachment/detachment assays. Magnesium and ferroussalts together with dilute agar, individually and all together, have noeffect on the sMMO activity of M. trichosporium OB3b.

F. Attached Cell-Column Longevity and Field Studies

Ultimately, the importance of this invention rests in the fact that thelongevity of the microbial filter is extended to practical andeconomical time-periods. Therefore, further laboratory studies wereperformed to validate that the improved attachment/detachment propertiesof the rosette isolate, in combination with additives, actually resultin the enhanced longevity of the microbial filter.

Because of its greatly elevated attachment and markedly lowereddetachment, when loaded in the presence of HPB buffer 1 mM MgCl₂ +100 μMFeSO₄ +0.025% agar, colony 5 and the above loading mixture were chosenfor an attached cell longevity experiment. Biodegradation profile datafor the 12 hour TCE-PR pulses that were delivered at time zero (i.e.,immediately after the 16 hour unattached cell washout step) and at fiveweekly time-points during the 15 week experiment are depicted in FIGS.8A, 8B, 8C, 8D, 8D, 8E, 8F, 8G, 8H, 8I, 8J, 8K, and 8L. During the TCEpulse times and the intervening periods the pump rate was consistentlymaintained at 0.4 mL/hour (1.25 cm/hour linear flow rate).

Over a 15-week period (almost 4 months) the average total amount of eachTCE-PR pulse was 9.2 nmoles (range 8.4-9.8 nmoles) and the columnflow-through residence time was ˜8 hours. The test and control columnswere run simultaneously and recovery of TCE from the control column wasalways at least 95%. For 8 weeks, the attached cell column filter almostcompletely degraded each 1250 ppb pulse of TCE (FIG. 8).

Weekly profiles of the non-metabolized TCE (A) and the bacterialdetachment (B) for the attached cell test column during the functionallongevity experiment in FIGS. 8A through 8L inclusive, are plotted inFIGS. 9A and 9B. It is noteworthy that, as seen in FIG. 9A, even after15 weeks, about 40% of the last TCE pulse was biodegraded.

FIG. 9A shows both the highest and average concentrations ofnon-metabolized TCE eluting from the attached cell test column duringeach of the ˜250 ppb TCE pulses over the course of the 15-weekexperiment. Elution of very small amounts (maximal ˜10 ppb or less) ofnon-metabolized TCE from the attached cell test column during the first8 weeks appeared to be slightly delayed, elution volumes 9-11 mL versusthe typical elution peak position of 5-8 mL for TCE from the minus cellcontrol column (FIG. 8). In this experimental set-up, this may be due tomicroscopic amounts of drying along the upper edge of the horizontalcolumn, which might have allowed the TCE to be retained longer in tinygas bubbles along this edge of the column. Any TCE in such bubbles wouldbe less bioavailable to the attached bacteria than the bulk of thedissolved TCE pulse which passed through the aqueous pore spaces of thesaturated sand. Irrespective, for the first 8 weeks, this resting cellfilter functioned extremely well, lowering the emerging average TCElevels to <5 ppb. The federal and state mandated regulatory level forthe complete remediation of TCE is 5 ppb. After 8 weeks the amount ofnon-metabolized TCE began to increase slowly in a nearly linear fashionup to 15 weeks.

Over the course of the column longevity experiment small fractions ofthe originally emplaced bacteria were continuously detaching during theconstant 10 mM HPB flow (FIG. 9B). After 15 weeks, extrusion of the sandand counts of the remaining cells indicated that only 34% of theinitially attached colony 5 cells were still on the column. Thedetachment half-life was ˜70 days (linear flow rate 1.25 cm/hour) inFIG. 9B in comparison to the 45-day half-life when the HPB flow rate was5 times faster (FIG. 9A) for colony 5 cells loaded in the presence of 1mM MgCl₂ +100 μM FeSO₄ +0.025% agar. A slower linear HPB flow of 1.25cm/hour was utilized in the column longevity experiment (FIG. 8) tomimic typical TCE-contaminated aquifer flows beneath the fieldcontaminated sites and to provide at least an 8 hour residence timewithin the attached cell filter.

Microscopic examination of cell population revealed that after the sandcolumn extrusion most of the remaining attached cells were M.trichosporium OB3b and that rosettes were still present, but at areduced level of ˜20% of the population (data not shown). The whole-cell[1,2-¹⁴ C]TCE degrading specific activity, had dropped from 52.3nmol/min/mg of dry cell wt to 4.3 nmol/min/mg (with formate) and from30.5 nmol/min/mg dry cell wt to 0.5 nmol/min/mg dry cell wt (minusformate). Interestingly, however, at 15 weeks with only 34% of theinitially emplaced bacteria still attached and with the cells remainingdisplaying 2% of their initial minus-formate whole-cell activity, it wasremarkable that the column filter still metabolized 40% of the ˜250 ppbTCE pulse at this time point (FIG. 9A).

The addition of MgCl₂, FeSO₄, and dilute agar to the loading bufferallowed not only to produce initially a filter with a much largerpopulation of attached cells (-1.5×10⁹ /g sand), but also to retain 34%of the cells on the filter by the end of the column longevity experimentafter 15 weeks (FIG. 9B). A calculation based on the number of cellsextruded, gives an estimated 6.2×10⁹ total bacteria, or 5.0×10⁸ /g ofdry sand, remaining on the column at the end of the experiment. Clearlythe initial attachment density was great enough and the detachmentsufficiently slow, that attachment/detachment processes under theseconditions (column linear flow rate of 1.25 cm/h) were no longer thecritical factors controlling the functional longevity of the simulatedfilter.

The longevity studies, the results of which are seen in FIGS. 8A through8L inclusive, were designed so that the biotransformation capacity ofthe bacteria for TCE would not be a limiting parameter. The sum totalamount of TCE in the 15 pulses of the longevity experiment (FIGS. 8 and9) was restricted to only 138 nmoles or 18 μg. The biomass initiallyattached to the 1-cm×10-cm sand column was ˜18.2×10⁹ cells or ˜10 mg ofdry cell wt. Thus, this amount of bacteria/mass initially could havebiodegraded ˜2.5 mg of TCE. Moveover, using ˜6 mg as the averageattached biomass over the course of the experiment illustrated in FIG.7B and allowing a ˜50-fold loss in the biotransformation capacity, basedon the formate rate data after 15 weeks, 6 mg of such cellstheoretically still could have degraded a total of ˜300 μg of TCE, i.e.,if given a much longer contact or residence time within the filter bygreatly slowing the TCE-pulse flow rate through the attached cell testcolumn.

The column longevity experiment (FIG. 8) was designed to be an extensionof previous 1.1-m flow-through experiments in a two-dimensional test bedthat demonstrated the short term effectiveness of the microbial filterapproach to degrade ˜100 ppb pulses of TCE. This was an HPB-saturatedOklahoma No.1 sand system in which a 10-cm thick M. trichosporium OB3bfilter was established. The 1-cm×10-cm glass columns were utilized torepresent cylindrical segments of that 10-cm thick test bed filter. Byusing flow rates that are common to chlorinated Voc contaminated sites,the functional longevity of an attached resting-cell population underconditions similar to those expected for a sand-filled trench in thefield can be reasonably estimated and it can be determined whetherattachment/detachment or whole-cell sMMO stability is the most likelylimiting factor for the long term operation of such a filter.

For the above reasons, the sand columns in FIG. 8 purposely were not fedTCE continuously as would be the case for an emplaced microbial filterin the field. Thus over the 8 weeks in which the ˜250 ppb pulses (12hours, 0.4 mL/h) of TCE were almost completely degraded by the attachedcell column, only ˜15 pore volumes of contaminated water actually weretreated. In a continuous contaminant input mode, however, at the flowrate employed ˜168 total pore volumes of TCE would have been treated.This translates to a significant length of contaminated flowinggroundwater, namely ˜17 meters over ˜2 months. For a more rigoroussimulation of the microbial filter concept and field implementationdiagramed in FIGS. 1 and 2, the apparatus in the illlustration (FIG. 3)can readily be adapted to run functional longevity tests in a continuousTCE (or other contaminant) mode. The continuous time can be varied froma few days to weeks. This was demonstrated using the parental strain ofM. trichosporium OB3b and employing site specific sediment andgroundwater. 50-mL syringes were used as the reservoir for contaminatedgroundwater and biodegradation was followed over a 72 hour time period(Third International In Situ and On-Site Bioreclamation Symposium BookSeries: Bioaugmentation for Site Remediation, 3: 15-29, 1995).

Analyses of the freshly grown bacteria at the start of the longevityexperiment and again after their extrusion from the sand column indicatethat most of the initial TCE degradation activity disappeared by 15weeks. The absolute degradation rate of TCE by the attached bacteria onthe column would be very much slower because 250 ppb is far below theapparent whole-cell Michaelis constant, 13.6 ppm, that was obtainedearlier for fresh bioreactor cultivated M. trichosporium OB3b cells.

A field test of the parental strain of M. trichosporium OB3b showed thatfilter could be formed in the subsurface. During the first 50 hours ofthe extraction phase, the filter performed very well. It removed 98% ofthe 425 ppb TCE. After that time, the filter degraded rapidly over thecourse of the 40-day field test (Environ. Sci. Technol., 30, 1982-1989,(1996). The decline in degradation was caused in part by reducedbacterial attachment with tie. This demonstrates the importance ofattachment/detachment parameters to overall field site success and theneed for this invention.

The functional longevity of a column biofilter, formed withresting-state rosette cells in the presence of the above additives, wasdetermined by challenging it with weekly 12-hour, ˜250 ppb pulses ofTCE. These sand-attached cells degraded the TCE pulses to <5 ppb for 8weeks. Some TCE breakthrough was apparent at the ninth week. After 15weeks, the remaining attached bacteria were still able to degrade 40% ofthe TCE pulse even though, when removed from the column, they retained<10% of their original rate of TCE-degrading activity.

Column results indicate that the operational longevity of the proposedsand-trench microbial filter for low TCE concentrations depends chieflyon the endogenous stability of the whole-cell soluble methanemonooxygenase system and that such a filter will need replenishment atapproximately 8-15 week intervals.

UTILITY

A variety of subsurface aquifer contaminants are treatable viabioremediation using a microbial filter approach.

Five different types of contaminants currently are known to be readilysusceptible to bacterial biodegradation or biotransformation. Petroleumand coal-derived hydrocarbons and their derivatives, halogenatedaliphatics including trichloroethene (TCE), halogenated aromatic andnitroaromatics, may be transformed by either aerobic or anaerobicbacterial processes according to this invention. A critical factor indeciding whether bioremediation is possible for a contaminant at aspecific site, is whether the microorganism to be used is compatiblewith the specific conditions and/or characteristics, such as pH,dissolved oxygen, soil type, etc. of the site.

This invention represents the first time that anyone has shown that arosette-forming methanotroph can be selected, cultured, itsattachment/detachment properties changed, and its longevity increased.As a result the methanotroph can, in this way, be utilizedadvantageously for specific remediation applications.

EXAMPLE 1 Culture Conditions

This example describes conditions used for culturing M. trichosporiumOB3b single cells or rosette clusters populations.

M. trichosporium OB3b was obtained from Professor R. S. Hanson (GrayFreshwater Biological Institute, University of Minnesota).

M. trichosporium OB3b was routinely maintained by suspension culturingat 30° C. on Higgins' nitrate minimal salts medium lacking Cu (NMS)according to Biotechnol. Bioeng., 38:423-433 (1991) under a methane/airgas mixture (1:1, v/v). After several months of continuous shake-flaskcultivation, cells from single well separated colonies were isolated bydilution-spreading on 1.7% agar plates containing NMS. The plates hadbeen incubated for 14-21 days at 30° C. in a gas-tight jar under a 1:1(v/v) methane/air gas mixture. These isolates were classifiedmicroscopically based on the presence of mainly single-cell or rosette(J. Gen. Microbiol., 61:205-218 (1970)) containing populations. Of tencolonies examined, an isolate with only single-cells and another isolatewith the largest percentage of rosette clusters were selected forfurther study. The two selected isolates maintained their single-cell orrosette character upon repeated suspension subcultured in shake-flasksor in a 5-L bioreactor (Bioflo III), commercially available, NewBrunswick Scientific, N.J. on Higgins nitrate minimal salts mediumlacking copper modified as reported previously in Hydro Sci. J. (IAHS),38: 323-342 (1993). The Modified Higgins' Salts (MHS) medium lacking Cuinitially contained 2× nitrate, 2× FeSO₄ --7H₂ O, and 40× Na₂ MoO₄ --2H₂O versus the NMS, and NiCl₂ was added at 7.5 μM. The bacteria wereharvested after 88-105 hours by centrifugation at 12,000×g, washed twotimes with 10 mM Higgins' medium phosphate buffer, pH 7.0 (HPB), andthen resuspended in the same buffer and adjusted to 2×10⁹ cells/mL usingan electronic particle counter mode ZB1 commercially available fromCoulter Electronics, Inc., Fla., equipped with a 30 μm aperture. Twicefiltered 4% NaCl was used as the counting solution according to WaterResour. Res., 30: 25-35 (1994).

EXAMPLE 2 Column Cell Attachment/Detachment Assays

This example describes column assays used for determination of cellattachment and cell detachment.

Cell attachment assays were carried out by a modification of thedisposable Pasteur-pipet column described in Water Resour. Res., supra.In a modified assay, sand columns were prepared in 1-cm (I.D.)×10-cmglass tubes (C-10 columns), commercially available from PharmaciaBiotech Inc., N.J. These columns contained ˜12 g of Oklahoma No. 1 sandobtained from T&S Materials, Gainesville, Tex., an ortho-quartziticsilicate with 84% of the grains having a diameter between 0.1 mm and0.25 mm, a bulk permeability of ˜8.5 darcys, and a porosity of 0.32±0.02in HPB. A 5-mL pipet was used to add the sand, as a slurry in 10 mM HPB.The sand was allowed to settle through a layer of HPB within the C-10tubes and continuous additions were made until the columns werecompletely filled. They were then washed with HPB for 3 hours at 2mL/min.

In the cell loading step, bacteria (2×10⁹ mL) were pulled onto thesaturated sand columns at 0.33 mL/min (63 cm/h column linear flow rate)for 2 hours with a peristaltic pump (8 channel, Minipulse III) obtainedfrom Gilson Medical Electronics, Inc., at 210C. The loading step wasfollowed by a washout step, in which the unattached bacteria wereremoved at 0.11 mL/min (21 cm/hour column linear flow rate) for 16hours. Column linear flow rates were determined experimentally from thebreakthrough times, i.e. the midpoints of the ascending limb profiles,given by short pulses of dilute phenol red (PR). Phenol red is a commoncolored pH indicator that can be quantified spectrophotometrically andwas previously validated to be a useful non-binding, inert tracer foraqueous flows through saturated sand. Water Resour. Res., 30: 25-35(1994). For the initial attachment measurements, the sand and attachedbacteria in the columns were then extruded into 25 mL graduatedcylinders and the volume was brought to 25 mL with distilled water.Cells were separated from the sand by shaking for 10 seconds andallowing the sand to settle for ˜1 min. The bacteria were thenenumerated with a Coulter counter.

For the detachment experiments, the connector tubing and the position ofthe peristaltic pump were switched after the 2-hour cell-loading step,so that the buffer could be pumped onto the tops of the columns andfractions collected from the 5 bottoms. Following the 16-hour washoutstep, the columns were rotated 90° to a horizontal position and the 10mM HPB flow rate was reduced to 0.033 mL/min (6.3 cm/hour column linearflow rate). This flow rate was selected to match the fastest knownlinear-flow rates for subsurface groundwater tested. Fractions werecollected every 24 hours and the detached bacteria were monitored with aCoulter counter.

EXAMPLE 3 Functional Longevity Study

This example describes studies performed for determination of microbialfunctional longevity.

Bacteria were suspended in 10 mM HPB containing 1 mM MgCl₂ +100 μM FeSO₄+0.025% agar obtained from Purified Agar, Oxoid Limited, (Basingstoke,Hampshire, England), at 2×10⁹ cells/mL and loaded onto a 1-cm×10-cmcolumn for 2 hours at 0.33 mL/min (Attached Cell Test Column). As acontrol, a similar column was loaded with the same buffer mixturelacking bacteria (Minus Cell Control Column). After the standard 16-hourwashout phase, the columns were turned horizontally and attached to asyringe pump (pump 22) obtained from Harvard Apparatus. Ten mL gas-tightHamilton syringes were filled with 10 mM HPB containing 110 μM PR and ˜2AM (˜250 ppb) trichloroethylene (TCE). Phenol red (PR) was included asan internal visual pH indicator and quantifiable conservative tracer forthe TCE pulse. Pulses were delivered via the pump for 12 hours at 0.4mL/hour (column linear flow rate of 1.25 cm/hour). Fractions (1.0 mL)were collected every 2.5 hours using 90°-bent stainless steel cannulasinserted into 5 mL vials sealed with open-top-closure screw caps andPTFE-faced red rubber septa. The PTFE findings were placed downwardtowards the gas phase and exactly 1 mL of air was removed from the vialsprior to collecting each sand-column fraction to prevent a subsequent20% pressure rise when the 1.0 mL liquid fractions entered these sealed5 mL vials. Tribasic sodium phosphate, 15 μmoles in 50 μL, also wasadded to the 5 mL vials prior to collecting the fractions in order toeliminate any residual TCE metabolism due to cell spillage from the sandcolumn into the collection vials. After 12 hours, the syringes werereplaced with identical syringes containing 10 mM HPB alone, andfractions were collected for an additional 20.5 hours at the sameflow-through rate. After 32.5 hours the syringe pump was replaced with aperistaltic pump and a flow of 10 mM HPB was continued at 0.4 mL/hourfor 5.65 days, i.e., until the start of the next TCE pulse. The columnswere rotated 180° along their horizontal axes every day to minimize anydrying along their top edges. A concern about regularly eliminating anypossible minute dry spots was driven by the fact that a few tiny bubblesperiodically formed in the columns, even under the most careful handlingconditions, when the input 10 mL syringes were switched. Each week thecolumns were challenged with an additional TCE-PR pulse in the samemanner.

Over the 5.65 day intervals between each successive TCE-PR pulse,fractions (10 mL each) were collected every 24 hours from the test andthe minus cell control columns. The detached cells in these fractionsand those in the 1.0 mL fractions collected during the TCE-PR pulsesalso were enumerated with a Coulter electronic particle counter.Bacterial spillage from the Test Column was determined by subtractingthe Control Column particle counts from the Test Column counts. Thispresented no problem in accurately assessing cell detachment overtimefrom the Test Column versus the Control Column because over the 15 weekexperimental period the former were in the range of 2.3×10⁷ to 1.4×10⁷while the later were much lower and nearly constant (1.6×10⁶ to 0.7×10⁶). After 15 weeks the columns were extruded, the cells were counted, anda total initial cell load was calculated.

EXAMPLE 4 Analytical Methods

This example describes analytical methods used for the development andtesting of this invention.

TCE was quantified by manually injecting 400 μL gas phase samples fromthe 5 mL vials into a gas chromatograph (Hewlett Packard model HP5890)equipped with a flame-ionization detector and a 6-ft stainless-steelpacked column (0.1% AT-1000 on Graphac-GC obtained from Alltech.Duplicate head-space samples were always analyzed for each vial. Thecolumn temperature was 135° C. and nitrogen was used as the carrier gasat a flow rate of 40 mL/min. The vial headspace TCE GC peak areas weredirectly compared to those generated with 5 mL vials to which 0.05 to1.0 nmole of TCE was added to vials also containing 1.0 mL of 10 mM HPBat 21° C., followed by equilibration and head-space sampling in anidentical manner. The GC detection limit for TCE (total in the vial) was<0.04 nmoles, when 400 μL head-space samples were withdrawn from thevials and analyzed. The GC area precisions for the TCE in thesand-column fractions and the standards were consistently ±2% and thefrequently checked gas-phase/aqueous-phase partitioning at 21° C. for˜250 ppb TCE within the 5 mL vials was 0.79.

Phenol red (PR) in the sand-column fractions was measured by diluting0.25 mL aqueous samples to 1.0 mL with 13.3 mM NAOH and then reading thepH 12 absorbance at 558 nm in a Model 260 spectrophotometer commerciallyobtained from Gilford Instrument Lab., Inc.

EXAMPLE 5 Detection of Whole-Cell sMMO Activity with TCE

This example describes detection of the cell sMMO activity with TCE.

At the time of harvest and again after the sand-column extrusion at 15weeks, the bacteria were assayed at 30° C. for their whole-cell specificrate of TCE degradation using a [1, 2⁻¹⁴ C) TCE (2,000 cpm/nmol)radiotracer assay, plus and minus 20 mM formate as a source of sMMOreducing power. (Hydro. Sci. J., 38: 323-342, (1993); ThirdInternational In Situ and On-Site Bioreclamation Symposium Book Series:Bioaugmentation for Site Remediation, 3: 15-29, (1995)). The initialsteady-state rate of biotransformation of TCE was 52.3 nmol/min/mg inthe presence of formate and 30.5 nmol/min/mg in its absence. (1,2⁻¹⁴ C]TCE 6,2 μCi/μmol) dissolved in water (or 0.1 mM HCl) was purchased fromthe Sigma Chemical Co. Previously, it was determined that this product,which is marketed as being ˜98% radiopure, typically contains ˜10%(range 4-20%) of storage-radiolytic contaminants. They arewater-soluble, non-volatile (stable to lyophilization) and consist of[¹⁴ C] bicarbonate, [¹⁴ C] formate, and other components that do notappear in a typical gas chromatographic profile for TCE. RecentlyEnviron. Sci. Technol., 29: 1210-1214 (1995) reported that [1,2⁻¹⁴ C]TCE, purchased from Sigma, volatilizes less rapidly than unlabeled TCEand suggested caution in its use, due to possible contaminants. Thiscontaminant problem was readily eliminated by first adjusting the Sigma[1,2⁻¹⁴ C] TCE with cold TCE to a concentration of 5 mM at the desiredspecific radioactivity and then carrying out a microdistillation.Aliquots (1.0 mL) of the above 5 mM [1,2⁻¹⁴ C] TCE were distilled at35-40° C. for 90 minutes from a glass sealed 5.0 mL vial, through asmall glass connector (Wheaton Claisen adapter) into a sealed 1.0 mLglass collection vial bathed in liquid nitrogen performed in dim lightbecause TCE vapors are somewhat light sensitive. The trapped [1,2⁻¹⁴ C]TCE was then reconstituted by injecting 0.1 mM HCl into the frozencollection vial and subsequently thawing its contents slowly. Overallrecoveries of actual (1,2⁻¹⁴ C] TCE were 70-80%.

The amount of dry cell weight was determined routinely from theabsorbance of the bacteria at 660 nm in the Gilford spectrophotometer.For both the single-cell and the rosette containing isolates of M.trichosporium OB3b, one unit of absorbance corresponded to 0.27 mg ofdry cell wt/mL.

EXAMPLE 6 Bioremediation of Sites Contaminated with ChlorinatedHydrocarbons

This example describes a method used in field bioremediation of theinvention. The system for field testing the invention is illustrated inFIGS. 1 and 2.

Bacteria were grown on a large scale in surface bioreactors, harvested,and taken to the site as a paste. They were resuspended in Higgin'sphosphate buffer alone. They were then injected into the subsurfaceusing several different configurations, as depicted in FIG. 2.

A Huff-And-Puff field test (Environ. Sci. Technol., 30: 1982-1989(1996)) was conducted with the parental and rosette cluster strain of M.trichosporium OB3b.

In these field tests, parental strain is separated from the rosetteisolate and both are emplaced using an unsupplemented loading buffer orthe buffer supplemented with additives. The filter consisting of theparental strain works successfully for only 2 days when unsupplementedand about 4 days when supplemented, while the rosette cluster isolateworks efficiently for over 8 weeks.

The laboratory test performed with rosette clusters confirmed theseresults. They did show, that an emplaced in situ biofilter workedsuccessfully for more than 8 weeks.

What is claimed is:
 1. A system for in situ bioremediation ofcontaminated water, said system comprising components:(a) a surfacebioreactor for growth of sufficient biomass of a selected stableisolate; (b) a mixing chamber wherein the isolate biomass is suspendedin water and additives are added; (c) an injection apparatus for amicrobial biofilter formation in situ by injecting the biomasssuspension of step (c); (d) the microbial biofilter emplaced in situ;(e) a means for extraction of water through the biofilter.
 2. The systemof claim 1 wherein the bioreactor further contains a means forcontrolling the temperature, pH, nutrients supply and oxygen or othergas needed for bacterial growth.
 3. The system of claim 2 wherein themeans for cell isolate separation is a centrifuge or a separationchamber.
 4. The system of claim 3 wherein the injection apparatus is aninjection pump and the cell suspension is injected through existinginjection or extraction wells into the aquifer to form the emplacedattached biofilter.
 5. The system of claim 4 wherein the biofilter hasthe density and thickness to assure a half-life longevity andfunctionality of the biofilter for at least about 8 weeks.
 6. The systemof claim 5 wherein the extraction means is an extraction pump or anextraction well.
 7. The system of claim 5 further comprising a means formonitoring a purity of the remediated water.
 8. The system of claim 7wherein the purity of decontaminated water is determined using amonitoring well or a laboratory equipment able to detect thecontaminant.
 9. A system for in situ bioremediation of water comprisingat least one contaminant, said system comprising components:(a) asurface bioreactor for growth of sufficient biomass of a selected stableisolate of a bacterial strain, said isolate having more pronouncedmetabolic characteristics in connection with said contaminant than ageneral pure strain of said bacterial strain; (b) a mixing chamberwherein the isolate biomass is suspended in water and additives areadded; (c) an injection apparatus for a microbial biofilter formation insitu by injecting the biomass suspension of step (c); (d) the microbialbiofilter emplaced in situ; (e) a means for extraction of water throughthe biofilter.
 10. The system of claim 9 wherein the bioreactor furthercontains a means for controlling the temperature, pH, nutrients supplyand oxygen or other gas needed for bacterial growth.
 11. The system ofclaim 10 wherein the means for cell isolate separation is a centrifugeor a separation chamber.
 12. The system of claim 11 wherein theinjection apparatus is an injection pump and the cell suspension isinjected through existing injection or extraction wells into the aquiferto form the emplaced attached biofilter.
 13. The system of claim 12wherein the biofilter has the density and thickness to assure ahalf-life longevity and functionality of the biofilter for at leastabout 8 weeks.
 14. The system of claim 13 wherein the extraction meansis an extraction pump or an extraction well.
 15. The system of claim 13further comprising a means for monitoring a purity of the remediatedwater.
 16. The system of claim 15 the purity of decontaminated water isdetermined using a monitoring well or a laboratory equipment able todetect the contaminant.
 17. The system of claim 9 wherein said isolatehas rosette-forming characteristics.
 18. A system for in situbioremediation of water comprising at least one contaminant, said systemcomprising components:(a) a surface bioreactor for growth of sufficientbiomass of a selected stable isolate of a bacterial strain, said isolatehaving rosette-forming characteristics and being able to metabolicallybiodegrade a water contaminant; (b) a mixing chamber wherein the isolatebiomass is suspended in water and additives are added; (c) an injectionapparatus for a microbial biofilter formation in situ by injecting thebiomass suspension of step (c); (d) the microbial biofilter emplaced insitu; (e) a means for extraction of water through the biofilter.
 19. Thesystem of claim 18 wherein the bacterial strain possesses oxygenase ormonooxygenase system.
 20. The system of claim 19 wherein the enzyme ismethane monooxygenase and bacteria is methanotrophic.
 21. The system ofclaim 20 wherein the bacteria is Methylosinus trichosporium OB3b and thestable isolate is a rosette cluster forming isolate of Methylosinustrichosporium OB3b.
 22. The system of claim 21 wherein the contaminantis a hydrocarbon.
 23. The system of claim 22 wherein the isolate ispregrown on nitrate minimal salt medium lacking copper to a cell biomasshaving a density necessary to biodegrade the contaminant for at least 8weeks.
 24. The system of claim 23 wherein the cell biomass is emplacedas the biofilter in an aquifer by suspending the cell biomass in aloading buffer or injection medium substituted with additives.
 25. Thesystem of claim 24 wherein the loading buffer or the injection medium issupplemented with magnesium salts, ferrous salts and with agar.
 26. Thesystem of claim 25 wherein the agar is added in concentration from about0.0075% to about 0.025% on a dry weight/0.01 M phosphate buffer (HPB)volume basis.
 27. The system of claim 18 wherein said rosette-formingcharacteristics comprise properties for increased attachment to thebiofilter compared to the same properties of said general pure strain.28. The system of claim 18 wherein said rosette-forming characteristicscomprise properties for a decreased rate of detachment from thebiofilter compared to the same properties of said general pure strain.